RESUMO
Microbial communities play a significant role in maintaining ecosystems in a healthy homeostasis. Presently, in the human gastrointestinal tract, there are certain taxonomic groups of importance, though there is no single species that plays a keystone role. Bacteroides spp. are known to be major players in the maintenance of eubiosis in the human gastrointestinal tract. Here we review the critical role that Bacteroides play in the human gut, their potential pathogenic role outside of the gut, and their various methods of adapting to the environment, with a focus on data for B. fragilis and B. thetaiotaomicron. Bacteroides are anaerobic non-sporing Gram negative organisms that are also resistant to bile acids, generally thriving in the gut and having a beneficial relationship with the host. While they are generally commensal organisms, some Bacteroides spp. can be opportunistic pathogens in scenarios of GI disease, trauma, cancer, or GI surgery, and cause infection, most commonly intra-abdominal infection. B. fragilis can develop antimicrobial resistance through multiple mechanisms in large part due to its plasticity and fluid genome. Bacteroidota (formerly, Bacteroidetes) have a very broad metabolic potential in the GI microbiota and can rapidly adapt their carbohydrate metabolism to the available nutrients. Gastrointestinal Bacteroidota species produce short-chain fatty acids such as succinate, acetate, butyrate, and occasionally propionate, as the major end-products, which have wide-ranging and many beneficial influences on the host. Bacteroidota, via bile acid metabolism, also play a role in in colonization-resistance of other organisms, including Clostridioides difficile, and maintenance of gut integrity.
Assuntos
Microbioma Gastrointestinal , Microbiota , Humanos , Bacteroides/genética , Trato Gastrointestinal , Ácidos e Sais Biliares/farmacologiaRESUMO
INTRODUCTION: Clostridioides difficile is the leading cause of healthcare-associated infections in the USA, with an estimated 1 billion dollars in excess cost to the healthcare system annually. C. difficile infection (CDI) has high recurrence rate, up to 25% after first episode and up to 60% for succeeding episodes. Preliminary in vitro and in vivo studies indicate that alanyl-glutamine (AQ) may be beneficial in treating CDI by its effect on restoring intestinal integrity in the epithelial barrier, ameliorating inflammation and decreasing relapse. METHODS AND ANALYSIS: This study is a randomised, placebo-controlled, double-blind, phase II clinical trial. The trial is designed to determine optimal dose and safety of oral AQ at 4, 24 and 44 g doses administered daily for 10 days concurrent with standard treatment of non-severe or severe uncomplicated CDI in persons age 18 and older. The primary outcome of interest is CDI recurrence during 60 days post-treatment follow-up, with the secondary outcome of mortality during 60 days post-treatment follow-up. Exploratory analysis will be done to determine the impact of AQ supplementation on intestinal and systemic inflammation, as well as intestinal microbial and metabolic profiles. ETHICS AND DISSEMINATION: The study has received University of Virginia Institutional Review Board approval (HSR200046, Protocol v9, April 2023). Findings will be disseminated via conference presentations, lectures and peer-reviewed publications. TRIAL REGISTRATION NUMBER: NCT04305769.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Adolescente , Humanos , Ensaios Clínicos Fase II como Assunto , Infecções por Clostridium/tratamento farmacológico , Suplementos Nutricionais , Método Duplo-Cego , Inflamação , Recidiva Local de Neoplasia , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , AdultoRESUMO
Abstract Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle is necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.
RESUMO
Clostridioides difficile (C. difficile) produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. Pannexin-1 (Panx1) channels allows the passage of messenger molecules, such as adenosine triphosphate (ATP), which in turn activate the P2X7 receptors (P2X7R) that regulate inflammation and cell death in inflammatory bowel diseases. The aim of this study was to verify the effect of C. difficile infection (CDI) in the expression of Panx1 and P2X7R in intestinal tissues of mice, as well as their role in cell death and IL-6 expression induced by TcdA and TcdB in enteric glial cells (EGCs). Male C57BL/6 mice (8 weeks of age) were infected with C. difficile VPI10463, and the control group received only vehicle per gavage. After three days post-infection (p.i.), cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry. In vitro, EGCs (PK060399egfr) were challenged with TcdA or TcdB, in the presence or absence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079), and Panx1 and P2X7R expression, caspase-3/7 activity and phosphatidylserine binding to annexin-V, as well as IL-6 expression were assessed. CDI increased the levels of Panx1 in cecum and colon of mice compared to the control group. Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not IL-6 gene expression in TcdA and TcdB-challenged EGCs. P2X7 receptor antagonist accentually reduced caspase-3/7 activity, phosphatidylserine-annexin-V binding, and IL-6 gene expression in TcdA and TcdB-challenged EGCs. In conclusion, Panx1 is increased during CDI and plays an important role in the effects of C. difficile toxins in EGCs, participating in cell death induced by both toxins by promoting caspase-3/7 activation via P2X7R, which is also involved in IL-6 expression induced by both toxins.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Conexinas , Proteínas do Tecido Nervoso , Receptores Purinérgicos P2X7 , Animais , Anexinas , Toxinas Bacterianas/metabolismo , Caspase 3/metabolismo , Morte Celular , Conexinas/genética , Conexinas/metabolismo , Mediadores da Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Fosfatidilserinas , Receptores Purinérgicos P2X7/genéticaRESUMO
Increased risk of intestinal dysfunction has been reported in patients after Clostridioides difficile infection (CDI). Enteric glial cells (EGCs), a component of the enteric nervous system (ENS), contribute to gut homeostasis. Previous studies showed that adenosine receptors, A2A and A2B, modulate inflammation during CDI. However, it is unknown how these receptors can modulate the EGC response to the C. difficile toxins (TcdA and TcdB). We investigated the effects of these toxins on the expression of adenosine receptors in EGCs and the role of these receptors on toxin-induced EGC death. Rat EGCs line were incubated with TcdA or TcdB alone or in combination with adenosine analogues 1h prior to toxins challenge. After incubation, EGCs were collected to evaluate gene expression (adenosine receptors and proinflammatory markers) and cell death. In vivo, WT, A2A, and A2B KO mice were infected with C. difficile, euthanized on day 3 post-infection, and cecum tissue was processed. TcdA and TcdB increased A2A and A3 transcripts, as well as decreased A2B. A2A agonist, but not A2A antagonist, decreased apoptosis induced by TcdA and TcdB in EGCs. A2B blocker, but not A2B agonist, diminished apoptosis in EGCs challenged with both toxins. A3 agonist, but not A3 blocker, reduced apoptosis in EGCs challenged with TcdA and TcdB. Inhibition of protein kinase A (PKA) and CREB, both involved in the main signaling pathway driven by activation of adenosine receptors, decreased EGC apoptosis induced by both toxins. A2A agonist and A2B antagonist decreased S100B upregulation induced by C. difficile toxins in EGCs. In vivo, infected A2B KO mice, but not A2A, exhibited a decrease in cell death, including EGCs and enteric neuron loss, compared to infected WT mice, reduced intestinal damage and decreased IL-6 and S100B levels in cecum. Our findings indicate that upregulation of A2A and A3 and downregulation of A2B in EGCs and downregulation of A2B in intestinal tissues elicit a protective response against C. difficile toxins. Adenosine receptors appear to play a regulatory role in EGCs death and proinflammatory response induced by TcdA and TcdB, and thus may be potential targets of intervention to prevent post-CDI intestinal dysmotility.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Ratos , Camundongos , Animais , Toxinas Bacterianas/metabolismo , Clostridioides difficile/fisiologia , Proteínas de Bactérias/genética , Infecções por Clostridium/metabolismo , Apoptose , Neuroglia/metabolismo , Receptores Purinérgicos P1/metabolismoRESUMO
5-Fluorouracil (5-FU) is an anticancer agent whose main side effects include intestinal mucositis associated with intestinal motility alterations maybe due to an effect on the enteric nervous system (ENS), but the underlying mechanism remains unclear. In this report, we used an animal model to investigate the participation of the S100B/RAGE/NFκB pathway in intestinal mucositis and enteric neurotoxicity caused by 5-FU (450 mg/kg, IP, single dose). 5-FU induced intestinal damage observed by shortened villi, loss of crypt architecture and intense inflammatory cell infiltrate as well as increased GFAP and S100B co-expression and decreased HuC/D protein expression in the small intestine. Furthermore, 5-FU increased RAGE and NFκB NLS immunostaining in enteric neurons, associated with a significant increase in the nitrite/nitrate, IL-6 and TNF-α levels, iNOS expression and MDA accumulation in the small intestine. We provide evidence that 5-FU induces reactive gliosis and reduction of enteric neurons in a S100B/RAGE/NFκB-dependent manner, since pentamidine, a S100B inhibitor, prevented 5-FU-induced neuronal loss, enteric glia activation, intestinal inflammation, oxidative stress and histological injury.
Assuntos
Fluoruracila/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosite/patologia , NF-kappa B/metabolismo , Neuroglia/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Sistema Nervoso Entérico/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Mucosite/metabolismo , Neuroglia/patologia , Estresse Oxidativo/efeitos dos fármacos , Fator de Transcrição RelA/metabolismoRESUMO
Amixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 µM (NTZ was toxic above 10 µM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · µg/ml (30 mg/kg dose) to 328 h · µg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 µg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.
Assuntos
Antibacterianos/farmacocinética , Benzamidas/farmacocinética , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Tiazóis/farmacocinética , Animais , Antibacterianos/sangue , Antibacterianos/farmacologia , Área Sob a Curva , Benzamidas/sangue , Benzamidas/farmacologia , Disponibilidade Biológica , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cães , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Infecções por Helicobacter/sangue , Infecções por Helicobacter/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Microbiota/efeitos dos fármacos , Microbiota/fisiologia , Microssomos Hepáticos/efeitos dos fármacos , Piruvato Sintase/metabolismo , Ratos , Tiamina Pirofosfato/metabolismo , Tiazóis/sangue , Tiazóis/farmacologiaRESUMO
Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression.
Assuntos
Toxinas Bacterianas/toxicidade , Dipeptídeos/farmacologia , Enterotoxinas/toxicidade , Células Epiteliais/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glutamina/farmacologia , Mucosa Intestinal/enzimologia , Proteína rhoA de Ligação ao GTP/biossíntese , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Colite/tratamento farmacológico , Colite/metabolismo , Colite/patologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Células Epiteliais/patologia , Mucosa Intestinal/patologia , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , RatosRESUMO
BACKGROUND: Clostridium difficile infection (CDI) continues to cause significant morbidity and mortality among hospitalized patients. Early diagnosis, contact precautions, and prompt therapy are crucial to the control of the disease and its spread. This study aims to develop an electronic screening tool to help identify patients who are at risk of CDI. METHODS: Six variables associated with CDI including antibiotic usage, age, and admission from another facility were identified. Logistic regression was used to weigh the variables, and then a predictive model was devised to help identify which patients may be at risk for developing CDI. A retrospective review of 29,453 records of hospitalizations was conducted including 274 cases of C difficile toxin positive patients to retrieve data for the model. RESULTS: The final model resulted in an area under the curve of 0.929, which suggests that the electronic screening tool will be an accurate predictor of predisposition to the disease. Model testing suggests a positive relationship between the total weight or score and the probability of developing the disease. CONCLUSION: An electronic screening tool may be an effective tool to assist in the accurate and timely identification of patients who may be predisposed to CDI during hospitalization.
Assuntos
Infecções por Clostridium/epidemiologia , Infecções por Clostridium/prevenção & controle , Métodos Epidemiológicos , Programas de Rastreamento/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Causalidade , Feminino , Hospitais Comunitários , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Estudos Retrospectivos , Medição de RiscoRESUMO
Clostridium difficile toxins A (TcdA) and B (TcdB) induce a pronounced systemic and intestinal inflammatory response. A(2B) adenosine receptors (A(2B)ARs) are the predominant adenosine receptors in the intestinal epithelium. We investigated whether A(2B)ARs are upregulated in human intestinal cells by TcdA or TcdB and whether blockade of A(2B)ARs can ameliorate C. difficile TcdA-induced enteritis and alter the outcome of C. difficile infection (CDI). Adenosine receptor subtype (A(1), A(2A), A(2B), and A(3)) mRNAs were assayed in HCT-8 cells. Ileal loops from wild-type rabbits and mice and A(2B)AR(-/-) mice were treated with TcdA, with or without the selective A(2B)AR antagonist ATL692 or PSB1115. A murine model of CDI was used to determine the effect of A(2B)AR deletion or blockade with the orally available agent ATL801, on clinical outcome, histopathology and intestinal interleukin-6 (IL-6) expression from infection. TcdA and TcdB upregulated A(2B)AR gene expression in HCT-8 cells. ATL692 decreased TcdA-induced secretion and epithelial injury in rabbit ileum. Deletion of A(2B)ARs reduced secretion and histopathology in TcdA-challenged mouse ileum. Deletion or blockade of A(2B)ARs reduced histopathology, IL-6 expression, weight loss, diarrhea, and mortality in C. difficile-infected mice. A(2B)ARs mediate C. difficile toxin-induced enteritis and disease. Inhibition of A(2B)AR activation may be a potential strategy to limit morbidity and mortality from CDI.
Assuntos
Clostridioides difficile/patogenicidade , Colo , Enterocolite Pseudomembranosa , Receptor A2B de Adenosina/metabolismo , Animais , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Linhagem Celular Tumoral , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/farmacologia , Regulação da Expressão Gênica , Humanos , Íleo/microbiologia , Íleo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Receptor A2B de Adenosina/genéticaRESUMO
PURPOSE: Human Immunodeficiency Virus (HIV) protease inhibitors (PI) remain a crucial component of highly active therapy (HAART) and recently have been demonstrated to have potent antitumor effect on a wide variety of tumor cell lines. However, discontinuation of therapy is an important issue, which may be related to various side-effects, especially diarrhea. The aim of this study was to evaluate the effects of nelfinavir (NFV), an HIV PI, and of alanyl-glutamine (AQ) supplementation, on intestinal cell migration, proliferation, apoptosis and necrosis, using IEC-6 cells and on intestinal crypt depth, villus length, villus area, mitotic index and apoptosis in Swiss mice. METHODS: Migration was evaluated at 12 and 24 h after injury using a wound healing assay. Cellular proliferation was measured indirectly at 24 and 48 h using tetrazolium salt WST-1. Apoptosis and necrosis were measured by flow cytometry using the Annexin V assay. Intestinal morphometry and mitotic index in vivo were assessed following a seven-day treatment with 100 mg/kg of NFV, given orally. In vivo proliferation and apoptosis were evaluated by intestinal crypt mitotic index and immunohistochemistry, respectively. RESULTS: In vitro, AQ supplementation enhanced IEC-6 cell migration and proliferation, following challenge with NFV. In vivo, AQ increased intestinal villus length, villus area, crypt depth and cell proliferation and cell migration, following treatment with NFV. AQ did not decrease cell death induced by NFV both in vivo and in vitro. CONCLUSIONS: AQ supplementation is potentially beneficial in preventing the effects of PIs, such as NFV, in the intestinal tract.
Assuntos
Apoptose/efeitos dos fármacos , Dipeptídeos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Nelfinavir/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Intestinos/citologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Masculino , Camundongos , Necrose , Ratos , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: Intestinal mucositis is one of the major troublesome side effects of anticancer chemotherapy leading to poor patient compliance. In this study we addressed the role of the novel apolipoprotein E (ApoE) COG 133 mimetic peptide in 5-fluorouracil (5-FU)-challenged Swiss mice and IEC-6 cell monolayers. Experiments were also conducted in C57BL6J ApoE knock-out mice to assess the effects of apoE peptide treatment. METHODS: Experimental groups were as follows: unchallenged controls, 5-FU-challenged mice (450 mg/kg, i.p) with or without the ApoE peptide (0.3, 1, and 3 µM, given twice daily i.p. for 4 days). Mice were sacrificed 3 days after 5-FU challenge. Proximal small intestinal samples were harvested for molecular biology and histological processing. We conducted ELISA assays and RT-PCR to target IL-1ß, TNF-α, IL-10, iNOS, and myeloperoxidase (MPO) to assess intestinal inflammation. Cell death and NF-κB assays were also conducted in apoE knock-out mice. In our in vitro models, IEC-6 cells were exposed to 1 mM of 5-FU in glutamine free media with or without the ApoE peptide (0.02, 0.2, 2, 5, 10, and 20 µM). We investigated IEC-6 cell proliferation and migration, 24 h after the 5-FU challenge. Additionally, apoptotic IEC-6 cells were measured by Tunel and flow cytometry. Equimolar doses of the ApoA-I (D4-F) peptide were also used in some experiments for comparative studies. RESULTS: Villus blunting and heavy inflammatory infiltrates were seen in the 5-FU-challenged group, findings that were partially ameliorated by the ApoE peptide. We found increased intestinal MPO and pro-inflammatory IL-1ß and TNF-α levels, and TNF-α and iNOS transcripts, and reduction of IL-10 following 5-FU treatment, each of which were partially abrogated by the peptide. Improvements were also found in IEC-6 cell apoptosis and migration following ApoE and D-4F treatment. CONCLUSION: Altogether, these findings suggest that the novel ApoE COG 133 mimetic peptide can reduce 5-FU-induced intestinal changes and potentially benefit mucositis.
Assuntos
Apolipoproteínas E/uso terapêutico , Fluoruracila/efeitos adversos , Enteropatias/induzido quimicamente , Enteropatias/tratamento farmacológico , Mucosite/induzido quimicamente , Mucosite/tratamento farmacológico , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/farmacologia , Apoptose/efeitos dos fármacos , Materiais Biomiméticos/farmacologia , Materiais Biomiméticos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Técnicas In Vitro , Enteropatias/patologia , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Jejuno/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosite/patologia , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Peroxidase/metabolismo , RatosRESUMO
BACKGROUND: Although several animal models of cryptosporidiosis have been reported, most involve genetically or pharmacologically immune-suppressed hosts. METHODS: We report challenge with excysted (in vitro and in vivo) and unexcysted (in vivo) Cryptosporidium parvum oocysts in human colonic adenocarcinoma (HCT-8) cells and weaned nourished and malnourished C57BL/6 mice, following outcomes of growth rate, stool shedding, and tissue burden. We tested treatment with an oligodeoxynucleotide containing unmethylated CpG motif (CpG-ODN) and alanyl-glutamine in vivo and in vitro. RESULTS: C. parvum-challenged mice showed prolonged weight loss (>10% over 4 days), robust stool shedding (>3 logs/d over 7 days), and epithelial infection in the ileum, cecum, and colon. Of 2 potential therapeutic compounds evaluated in the model, CpG-ODN reduced body weight loss (to <6% on days 3-7 after challenge), reduced shedding of organisms (by 25% on days 1 and 3 after challenge), and decreased the burden of parasites in the ileum. Alanyl-glutamine showed similar benefits. In vitro findings suggested that effects on the epithelial component of the mucosa probably likely responsible for beneficial effects seen in vivo. CONCLUSIONS: Weaned mice provide a convenient and reproducible model of cryptosporidial disease, including its vicious cycle with body weight loss and heavier infection with malnutrition, and this model may be useful in exploring innovative therapeutic solutions for this challenging infectious disease.
Assuntos
Criptosporidiose/terapia , Cryptosporidium parvum/patogenicidade , Desnutrição/parasitologia , Desnutrição/terapia , Animais , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/parasitologia , Colo/patologia , Criptosporidiose/complicações , Criptosporidiose/patologia , Cryptosporidium parvum/isolamento & purificação , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Dipeptídeos/uso terapêutico , Fezes/parasitologia , Feminino , Humanos , Íleo/efeitos dos fármacos , Íleo/parasitologia , Íleo/patologia , Desnutrição/complicações , Desnutrição/patologia , Camundongos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/uso terapêuticoRESUMO
BACKGROUND: Toxins A and B (TcdA and TcdB) are Clostridium difficile's principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure. RESULTS: We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis. CONCLUSIONS: This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Ceco/citologia , Ciclo Celular/efeitos dos fármacos , Enterotoxinas/toxicidade , Células Epiteliais/efeitos dos fármacos , Íleo/citologia , Transcrição Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Biologia de Sistemas/métodos , Fatores de TempoRESUMO
Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases.
Assuntos
Inibidores de Adenilil Ciclases , Antidiarreicos/química , Antidiarreicos/farmacologia , Diarreia/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Animais , Antidiarreicos/uso terapêutico , Toxinas Bacterianas/farmacologia , Linhagem Celular , Cricetinae , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , GMP Cíclico/antagonistas & inibidores , GMP Cíclico/biossíntese , Diarreia/enzimologia , Enterotoxinas/farmacologia , Inibidores Enzimáticos/uso terapêutico , Proteínas de Escherichia coli , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Ratos , Bibliotecas de Moléculas PequenasRESUMO
Cryptosporidium parvum is a leading pathogen in children in developing countries. To investigate whether early postnatal malnutrition leads to heavier C. parvum infections, we assessed intestinal adaptation and parasite load in suckling mice during the first 2 wk of life, analogous to the first postnatal yr in humans. Undernutrition was induced by daily C57BL6J pup separation from lactating dams. Half of the pups were separated daily, for 4 hr on day 4, 8 hr on day 5, and for 12 hr from day 6 until day 14. On day 6, each pup received an oral inoculum of 10(5) to 10(7) parasites in 10-25 microl of PBS. Littermate controls received PBS alone. Stools were assessed from days 8, 11, and 14 for oocyst counts. Mice were killed on day 14, 8 days postinoculation, at the peak of the infection. Ileal and colon segments were obtained for histology, real-time and reverse transcriptase PCR, and immunoassays. Villus and crypt lengths and cross-sectional areas were also measured. Undernourished and nourished mice infected with excysted 10(6) or 10(7) oocysts exhibited the poorest growth outcomes compared with their uninfected controls. Nourished 10(6)-infected mice had comparable weight decrements to uninfected undernourished mice. Body weight and villi were additively affected by malnutrition and cryptosporidiosis. Hyperplastic crypts and heavier inflammatory responses were found in the ilea of infected malnourished mice. Undernourished infected mice exhibited greater oocyst shedding, TNF-alpha and IFN-gamma intestinal levels, and mRNA expression compared to nourished mice infected with either 10(5) or 10(6) oocysts. Taken together, these findings show that Cryptosporidium infection can cause undernutrition and, conversely, that weanling undernutrition intensifies infection and mucosal damage.
Assuntos
Criptosporidiose/complicações , Desnutrição/complicações , Animais , Animais Lactentes , Colo/metabolismo , Colo/parasitologia , Colo/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Íleo/metabolismo , Íleo/parasitologia , Íleo/patologia , Interferon gama/análise , Interferon gama/genética , Camundongos , Camundongos Endogâmicos C57BL , Contagem de Ovos de Parasitas , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/genética , Desmame , Aumento de PesoRESUMO
In this study, we have examined the role of glutamine derivatives in reducing 5-fluorouracil (5-FU)-induced epithelial damage in an undifferentiated crypt intestinal cell line, IEC-6. In this model, we have investigated proliferation indirectly by detecting the enzyme-derived formazan dye from the tetrazolium salt WST-1 in viable cells at 24 and 48 h after 5-FU treatment. Migration was measured at 12 and 24 h after razor scraping of the cell monolayer. Cell death was measured by quantifying the percentage of apoptotic and necrotic figures by flow cytometry at 12 and 24 h following 5-FU challenge. Neither glutamine nor alanyl-glutamine prevented 5-FU-induced apoptosis and necrosis in IEC-6 cells at 12 and 24 h after 5-FU challenge. However, glutamine and alanyl-glutamine enhanced migration and proliferation when compared with 5-FU-treated controls (P < 0.05). These new findings support our earlier study on the benefit of oral glutamine in enhancing epithelial recovery after 5-FU challenge.